EXPERIMENTAL PARASITOLOGY 37, 373379

Experimental

( 1975)

Acute Babesia cafdli

Infections

II. Response of Platelets and Fibrinogen PATRICJA C. ALLEN, WAYNE United

M. FRERICHS,

States Department

Accepted

September

AND

A. A. HOLBROOKl

of Agriculture

11, 1974

ALLEN, P. C., FRERICHS, W. M., AND HOLBROOK, A. A. 1975. Experimental acute Babe& caballi infections. II. Response of platelets and fibrinogen. Experimental Parasitology 37, 373379. Ponies were acutely infected with Babesia caballi by inoculation with infected red blood cells ( RBCs) containing 1 X 10’ and 1 X 10’ piroplasms. A series of blood samples taken before and after inoculation were analyzed for platelets and fibrinogen, and the results compared with similar analyses made on challenged, premunized ponies and on equids inoculated with uninfected RBCs. In acutely infected animals there were immediate decreases in platelet counts that persisted at least through Day 18 after inoculation (-41). Concomitantly, plasma fibrinogen levels rose, reaching peak values between Days 6-17. Clot retractions in vitro were impaired in these ponies during Days 9-16. No large diminutions in platelet counts or elevations of fibrinogen levels were observed in the challenged, premunized ponies or the group transfused with uninfected RBCs. In fact, the effect of challenge was to maintain or increase platelet counts. Our results plainly indicate that B. caballi can elicit alterations in clotting factor levels in its hosts during acute infections. INDEX DESCRIPTORS: Babesia cabal& Ponies; Platelets; Fibrinogen; clottable thrombin; Clot retraction; Intravascular coagulation.

ports concerning factors that may be related to potential coagulation abnormalities in equine piroplasmosis. In our detailed study of experimental acute Babe& caballi infections, we have included measurements of platelets and fibrinogen as initial investigations into the possible roles that changes in levels of ‘clotting factors might play in the pathogenesis of this type of equine piroplasmosis. In order to differentiate a susceptible from an immune response and to determine any contribution from the transfused RBCs themselves to the effects observed, we have compared the data obtained from acutely infected animals with data from challenged, premunized ponies and from equids transfused with uninfected RBCs.

Coagulation abnormalities have been reported as complications of various hemoprotozoan diseases (Malherbe 1956; Dennis et al. 1966, 1967; Maegraith 1948; Neva et al. 1970; Sheagren et al. 1970; O’Leary et aL 1972; Butler et al. 1973). Reports on atypical symptomatology of Babesiu infections of cattle and dogs have referred to the presence of parasitic emboli, agglomeration of parasitized cells in capillaries, and petechiation of mucus membranes and internal membranes ( Malherbe 1956). Mahoney and Goodger (1969) have indicated the probable ,presence of fibrinfibrinogen split products in sera from cattle infected with Babesia argentiw. However, to our knowledge, there have been no re1 Animal Parasitology Institute, Agricultural Research Service, Beltsville, Maryland 20705. 373 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

374

ALLEN, FREBICHS AND HOLBROOK MATEFXALS AND METHODS

RESULTS

The animals, source of B. cab&, experimental groups, and blood collection schedules are identical to those described in paper I of this series (Allen et al. 1975). Briefly, Group 1 consisted of six ponies; three of which (subgroup A: 155, 154, and 152) were inoculated with 1 x lo” piroplasms at a rate of 2 ml packed cells (PC)/ kg body wt, and three of which (subgroup B: 153, 132, and 119) were inoculated with 1 x lo8 piroplasms (0.2 ml PC/kg). Group 2 consisted of three of the ponies (154, 153, and 119) that survived infection in Group 1. They were inoculated with 1 X 10’ piroplasms (1 ml PC/kg). Group 3 consisted of a horse (148), a burro (149), and a pony (162) sensitized to equine RBCs (had received blood from a Babes& carrier cleansed by drug treatment). These animals received uninfected RBCs from separate pony donors at a rate of 1 ml PC/kg body wt. Hematological determinations: Platelets in all blood samples (1.3 mg/ml EDTA used as anticoagulant) were counted directly in hemocytometers, according to Schalm ( 1965). Thrombin-clottable fibrinogen in oxalated plasma was determined by a modification of the method of Ratnoff and Menzie ( 1951), as described by Langdell ( 1969). Plasma protamine paracoagulation (PPP) tests for circulating fibrin monomer lcomplexcs (Seaman 1970) were performed on selected oxalated plasma samples from all test animals before and after inoculation. The test is as follows: Frotamine sulfate (0.1 ml of a 1% aqueous solution) is added with mixing to 1 ml oxalated plasma in a test tube. The tube is incubated at 37 C for 15 min. The test is positive with the formation of a flocculant precipitate. Mere cloudiness is considered negative. Clot retractions were qualitatively observed on blood samples taken for serum by noting the relative volume of serum retrievable from a lo-ml sample. This normally ranged between 5-7 ml.

Preinoculation platelet and fibrinogen values for Group 1 ponies, as well as preinoculation and postinoculation values for animals in Groups 2 and 3, were within reported normal ranges (platelets, l-6 X 105/ 1~00-500 mm3, Schalm 1965; fibrinogen, mg/lOO ml, Schalm 1970). In both subgroups of acutely infected ponies (Group 1) the platelet count decreased within 4 days after inoculation (AI) and remained low for at least 18 days AI. All acutely infected ponies except 152 exhibited maximum decreases in platelet counts between Days 5-16 AI (Fig. 1). Maximum decrease for 152 was on Day 20 AI, 2 days before death. The period 5-19 days Al encompassed which parasitemias the times during reached peak values (Fig. 1). Pony 155 died in this period. During the same time, the thrombin-clottable fibrinogen increnscd, reaching maximum values between Days 6-17 AI. Peak fibrinogen values approximated the range (1000-1200 mg/lOO ml) reported by Schalm (1970) for horses with severe bacterial infections. Clot retraction of whole blood samples from the acutely infected ponies was noticeably impaired during Days 9-16 AI, only 2-4 ml serum/l0 ml blood being retrieved, even after repeated centrifugation. No retraction problems were noticed in either of the other two groups. No analogous protracted decreases in platelet counts occurred in either Groups 2 or 3 (Fig. 2). There was a transitory decrease shown by Group 2 panics (challenged, premunized) on Day 1 AI. However, the overall effect of challenge inoculation was to maintain or increase the platclet count (Fig. 2). No changes in fibrinogen content were observed in Group 2 that were similar to changes in acutely infected ponies. However, the burro in Group 3 exhibited transitory increases in fibrinogen during Days 5-16 AI. The PPP test was not useful for deter-

R. CUbdi

INFECTION:

PLATELETS

375

AND FIBRINOGEN

Inoculated with IX108 piroplasms

Inoculated with I X 10’ piroplasms

800 700

Group IA

p.-__-..q 155 154 ---4 152

Group I8

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zoo 100

1200 E IO00 8 7 m 800 E

d 600 B g 400 L

D -r&d' 2oo $2

11111,,1111111111,1,,,,1,,,,~,~,,~,,,,,

5

IO

15

20

25

30

35

L

Days after inoculation

FIG. 1. Platelet counts and fibrinogen levels in ponies inoculated (Group 1A) and 1 X 10’ piroplasms (Group 1B) correlated with

mining the effect of B. cubnlli infections on the presence of fibrin monomer complexes in plasma because preinoculation and control samples, as well as postinoculation samples, gave positive tests. DISCUSSION

Very little has been published specifically describing alterations in blood clotting mechanisms elicited by babesial infections. However, reports on atypical symptomatology in bovine and canine infections

with 1 X lo0 piroplasms daily parasitemia counts.

(Malherbe 1956) suggest that derangements of the normal hemostatic processes may take place, and that they may constitute part of the pathogenic effects of such infections. Most of the knowledge concerning effects of hemoprotozoan infections on blood clotting has come from studies of human and simian malaria (Dennis et al. 1967; George et al. 1966; Sheagren et al. 1970; Neva et al. 1970; O’Leary et al. 1972; Butler et al. 1973). A consistent finding

376

ALLEN,

FRERICHS

Challenged with IX104 piroplosms

0

5

IO

15

20

25

AND

HOLBROOK

Transfused with uninfected erythrocytes

0

5

Days after inoculation FIG.

2. Platelet

piroplasms

(Group

counts and fibrinogen levels in premunized 2) and equids transfused with uninfected

has been that in such infections the platelets decrease in circulation, along with the other blood cells. There is some dispute about the cause of the observed thrombocytopenia. Some believe it is due to multiple clot formation (intravascular coagulation) per se (Dennis et al. 1966, 1967; O’Leary et al. 1972). Others emphasize immune damage to the platelets with subscquent phagocytosis by the reticuloendothelial system (RES) (Neva et al. 1970; Sheagren et al. 1970; Butler et al. 1973). However, these views may not be mutually exclusive. Our data show that the platelet decreases observed in acutely infected ponies (Group I) were directly related to the sudden presence of B. caballi in susceptible animals, and that the counts remained low while parasites were observed in the blood. Platelet decreases did not take place in

IO

15

20

25

30

Days after inoculotlon

ponies challenged RBCs (Group 3).

with

1 X 10’

premunized animals and were not a result of transfusion with packed red cells. It is possible that in B. cab& infections platelets are damaged by immune mechanisms (humoral) not operative in premunized animals. They may then form foci for thrombi, or may be phagocytized in large numbers by a stimulated RES. We have already indicated that the RES appears to be greatly stimulated in Group 1 ponies (Allen et al. 1975). Circulating fibrinogen levels in healthy animals result from an equilibrium established between synthesis by the liver parenchyma (Foreman and Barnhart 1964) and removal, which occurs in several steps: (1) conversion to fibrin clots by thrombin, (2) lysis of clots as well as fibrinogen by plasmin, (3) removal of lysis products and soluble fibrin by the RES (Barnhart and Cress 1967; Walsh and Barnhart 1969).

B. CUbUlli INFECTION:

PLATELETS AND FIBRINOGEN

High levels of circulating fibrinogen are known to occur following cellular damage due to inflammatory or necrotizing lesions (Losner and Volk 1965; Castle 1967; Goodale and Widmann 1969; Schahn 1970; Schalm et al. 1970). They are also known to result from an RES blockade (Beller 1969; Miiller-Berghaus 1969) and to be present in chronic intravascular coagulation (McKay 1973) in which they arise from a rebound ,phenomenon, that is, stimulation of fibrinogen synthesis at a greater than normal rate (Searcy 1969). The increases in fibrinogen and decreases in platelets which we have observed in Group 1 ponies approximate the pattern by which McKay (1973) characterizes chronic intravascular coagulation. He also suggests that chronic intravascular coagulation will lead to a hypercoagulable state which may upon a secondary clotting stimulus result in large vessel thrombosis. We have not observed the latter phenomenon in Group 1 ponies. However, we have observed that blood of splenectomized and intact burros infected with B. c&zZZi will form sludgy clots right at the time of collection even in the presence of EDTA. In fact, syringe needles have become clogged with clots (unpublished data). We had hoped to implicate further intravascular ‘coagulation in the .pathogenesis of B. cab& infections by demonstrating the presence of fibrin monomer complexes in infected plasma with the FPP test. Howand control ever, many preinoculation sampIes as well as postinoculation samples gave a positive test. These results suggest the prior existence of some vascular fibrin deposition (Seaman 1970) in these animals, possibly brought about by strongyles, common parasites of equids (Drudge et al. 1966). The combined effects of platelet decrease and fibrinogen increase were apparently manifest in Group 1 ponies by an impairment of whole blood ‘clot retraction. During Days 9-16 AI this was particularly

377

noticeable because of the diffi~culty in harvesting sufficient serum for our analyses. It is not known how impaired clot retraction as seen in uitm actually affects an animal in uiuo (Bowie and Owen 1971), but it would seem reasonable that longer than normal stasis of blood flow would occur in vessels where clots had formed. A review of the literature reveals that the normal hemostatic processes in the horse are quantitatively different from those in humans. Horses have been found to have normally longer clotting times ( Fantl and Mar-r 1958), longer prothrombin times, higher factors V, VII, and IX and lower factors XI and XII (Abildgaard and Link 1965). In addition, horses are reported to have higher fibrinolytic potential than humans (Messow 1959; Hampl 1968) although this seems at variance with observations that fibrinogen increases in a number of inflammatory equine diseases ( Schalm 1970; Schalm et al. 1970). Because of these differences it is difficult to judge pathogenic effects of altered ‘clotting factor levels in equids by human standards. Detailed coagulation studies of both healthy and infected equids need to be undertaken to define more precisely the role which altered clotting factor levels play in the pathogenesis of B. cabal& infections. Such studies are now in progress. ACKNOWLEDGMENTS The authors gratefuIIy acknowledge the technical assistanceof R. Ellis, D. Davis, J. Hoang, 0. McGaha, L. Owens, and G. Strachan. REFERENCES ABILDGAARD, C. F., AND LINK, R. P. 1965. Blood coagulation and hemostasis in #thoroughbred horses. Proceedings of the Society for Experimental Biology and MedZcine 119, 212-215. ALLEN, P. C., FRERICHS, W. M., AND HOLBROOK, A. A. 1975. Experimental acute Babesia caballi infections. I. Red blood cell dynamics. Experimental Pamsitology 37, 67-77. BARNHART, M. I., AND CRESS, D. C. 1967. Plasma clearance of products of fibrinolysis. In “Ad-

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FRERICHS

vances in Experimental Medicine and Biology” (N. R. Di Luzio and R. Paoletti, Eds.), pp. 492-502, Vol. 1. Plenum Press, N. Y. BELLER, F. K. 1969. The role of endotoxin in disseminated intravascular coagulation. ZXrombasis et Diathesis Haemorrhagica Supplement 36, 125-149. BOWIE, E. J. W., AND OVEN, C. A., JR. 1971. Clinical disorders related to blood platelets. In “The Circulating Platelet” (S. A. Johnson, Ed.), pp. 477-539. BUTLER, T., TONG, M. J., FLETCIIER, J. R., DOSTALEK, R. J., AND ROBBINS, T. 0. 1973. Blood coagulation studies in Plasmodium falciparum malaria. American Journal of the Medical Sciences 265, 63-67. CASTLE, W. B. 1967. Disorders of the blood and blood forming tissues. In “Pathologic Physiology” (W. A, Sodeman and W. A. Sodeman, Jr., Eds.), pp. 751-896. Saunders, Philadelphia. DENNIS, L. H., EICHELBERGER, J. W., JR., VON DOENHOFF, A. E., AND CONRAD, M. E. 1966. A coagulation defect and its treatment with heparin in Plasmodium kno&xi malaria in Rhesus monkeys. Military Medicine 131 (Supplement), 1107-1110. DENNIS, L. H., EICHELUERGEH, J. W., IE*TXIAN, M. M., AND CONRAD, M. E. 1967. Depletion of coagulation factors in drug-resistant Plasmodium falciparum malaria. Blood 29, 713721. DRUDGE, J. H., LYOSS, E. T., AND SZANTO, J. 1966. Pathogenesis of migrating stages of helminths with special reference to Strom&s uulgaris. In “Biology of Parasites” (E. J. L. Soulsby, Ed,), pp. 199-214. Academic Press, N.Y. FANTL, P., AND MARL, A. G. 1958. The coagulation of horse blood. JOUITU~ of Physiology 142, 197-207. FORTRAN, W. B., AND BARNHART, M. I. 1964. Cellular site for fibrinogen synthesis. Journal of the American Medical Association 187, 128-132. GEORGE, J. N., STOKES, E. F., WIKER, D. J., AND CONRAD, M. E. 1966. Studies of the mechanism of hemolysis in experimental malaria. Military Medicine 131( Supplement), 12171224. GOODALE, R., AND WIDXIANN, F. K. 1969. “Clinical Interpretation of Laboratory Tests” 6th ed. p. 93. F. A. Davis, Philadelphia. HAMPL, W. 1968. “Untersuchungen iiber die Blutgerinnung bei Pferd, Rind und Hund, Fibrinogen (quantitative), Fibrinogenzeit, Hitzefibrin, Fibrinolyse.” Veterinary Dissertations, Munich.

AND

HOLBROOK

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INFECTION:

PLATELETS

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AND FIBRINOGEN

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function in naturally acquired human malaria. ]ournal of Laboratory and Clinical Medicine 75,481487. WALSH, R. T., AND BARNHART, M. I. 1969. Clearance of coagulation and fibrinolysis products by the reticuloendothelial system. Thrombosis et Diathesis

83-97.

Haemorrhagica

Supplement

36,